The development of autoimmunity involves the failure of the mechanisms that regulate the ability to discriminate self from non-self. The primary means of regulating self-tolerance is through the deletion of self- reactive cells in the thymus. However, this mechanism is not perfect and auto-reactive clones do escape into the periphery. Peripheral tolerance is generated through a variety of mechanisms including T cell anergy and T cell indifference or ignorance. Recently, another, more active, mechanism of tolerance induction has been identified that is controlled by a population of regulatory T cells that actively suppress the function of auto- reactive T cells. These T cells, known as Treg cells, play a critical role in preventing responses to self-antigens as fatal autoimmunity develops in their absence. However, the mechanism by which these cells perform their tasks is as yet unclear. Recent work has shown that the forkhead/winged-helix protein FOXP3 is expressed predominantly in Treg cells, and is both necessary and sufficient for their development and function. FOXP3 acts as a transcriptional regulator, targeting cytokine genes whose expression is induced in stimulated CD4+ T cells. While the importance of FOXP3 to Treg development and function is clear, several important questions remain unanswered. The experiments in this proposal will address: 1. The mechanism of FOXP3-mediated transcriptional repression through a structure/function analysis of FOXP3 and characterization of FOXP3- interacting proteins;2. The role of FOXP3-ROR3t interactions in the control of Treg Th17 differentiation, as our recent data has shown that FOXP3 interacts with and inhibits ROR3t, and that the interactions between FOXP3 and RORgt determine the differentiative fate of CD4 T cells to the Treg or Th17 lineage;3. It is becoming clear that Tregs are recruited to sites of autoimmune inflammation, but that factors present at those site abrogate the ability of the Tregs to suppress the response. We will use a mouse model of organ-specific autoimmunity to indentify and characterize factors capable of inhibiting Treg function. PUBLIC HEALTH RELEVANCE: Identification and characterization of the factors that control the ability of the immune system to distinguish self from non-self is critical to the development of therapeutics for the treatment of autoimmune diseases. A population of T cells referred to as regulatory T cells, or Tregs, is responsible for regulating immune responses to self. The transcription factor FOXP3 has been found to be essential for their development and function, and the loss of FOXP3 leads to fatal autoimmunity in humans and mice. Thus, a better understanding of the mechanism by which FOXP3 controls Treg function will provide insight into Treg biology, and identify potential targets for manipulation of this important T cell subset.